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Toxin production by Aeromonas spp. from different sources.

One hundred and eleven isolates of Aeromonas from water and from human sources were identified to species level and tested for the production of cytotoxin. These results were correlated with the source of each isolate and, for those from human faeces, with the clinical history of diarrhoea. A. caviae predominated in water, comprising 16 of 32 isolates; only one isolate from water was A. sobria. In human faecal samples 21 of 76 isolates were A. sobria; this was a significant difference. Cytotoxin producing strains were significantly more common in patients with no known cause for their gut symptoms. It is concluded that gastro-enteritis caused by Aeromonas is related to species and to production of cytotoxin.

Aeromonas↗

Adhesion of clinical and environmental Aeromonas isolates to HEp-2 cells.

A total of 63 Aeromonas strains isolated from diarrhoeal faeces or water samples were tested for adhesion to HEp-2 cells. An association between diarrhoea and high level adhesion was observed in that 12 of the 34 faecal isolates and none of the 29 environmental isolates yielded greater than 20 bacteria per HEp-2 cell in the adhesion assay. The proportion of high adherers was significantly greater for A. sobria (57%) than for A. hydrophila isolates (19%). Three of the eight faecal A. caviae isolates were also found to be high adherers. All of the environmental isolates were heavily pilated with pili having a mean diameter of 5 nm and a mean length of 420 nm; these were termed type-S pili. Of the 34 faecal isolates, 32% possessed S pili, and 68% were lightly pilated with up to 15 thin, flexible type-L pili, of mean diameter 2.5 nm and mean length 960 nm. Type-L pilation was associated with a high level of HEp-2 cell adhesion, and was more common in A. sobria and A. caviae than in A. hydrophila isolates. These results suggest that adherence to HEp-2 cells is a useful model for the investigation of Aeromonas enteropathogenicity, and that adhesion may be pilus-mediated in this organism.

Aeromonas↗

Production of haemolysis and its correlation with enterotoxicity in Aeromonas spp.

A total of 147 clinical and environmental isolates of Aeromonas that included 14 A. hydrophila, 60 A. sobria and 73 A. caviae strains was tested for haemolysin production and its correlation with enterotoxicity; 108 isolates produced beta-haemolysis. For A. hydrophila and A. sobria, titres of haemolysin were 16-128 HU/ml and for A. caviae, 16-64 HU/ml. In the ileal loop test, 82 (55.8%) strains of Aeromonas spp. produced enterotoxin. Of the beta-haemolytic strains, 72.7% of A. hydrophila, 58.6% of A. sobria and 68.6% of A. caviae isolates caused fluid accumulation in rabbit ileal loops. One strain each of alpha-haemolytic A. sobria and A. caviae, one of non-haemolytic A. sobria and nine of non-haemolytic A. caviae also caused a secretory response. The beta-haemolytic strains caused significantly more (p < 0.05) fluid accumulation than the alpha- and non-haemolytic isolates regardless of their species designation. The remaining 65 (44.2%) isolates belonging to the three species included alpha-, beta- and non-haemolytic strains: they failed to cause fluid accumulation in the initial experiments but did so after one to three consecutive passages through rabbit ileal loops. Two alpha- and 13 non-haemolytic strains switched to production of beta-haemolysis when they showed positive ileal loop reactions. However, on repeated subcultures or on storage in the laboratory, all of them reverted to their original haemolytic character and no longer produced enterotoxic activity.

Adolescent↗

Cytotoxic and haemagglutinating activities of motile Aeromonas species.

Cytotoxic and haemagglutinating properties were determined in 114 Aeromonas strains isolated from various sites in slaughtered lambs and from processed lamb meat. Cytotoxic activity on Vero cells was observed in 48 (42%) of the strains. It was more common in A. sobria and A. hydrophila isolates than with A. caviae isolates. Haemagglutination (HA) activity was found frequently in motile aeromonads irrespective of species; it was present in 50% of A. sobria strains, 51% of A. hydrophila strains and 48% of A. caviae strains. HA was inhibited by fucose, galactose and mannose at low concentration, and in most cases, two or three of these sugars were inhibitory. A significant association was found between certain HA-inhibition patterns and the production of cytotoxin by Aeromonas spp.

Aeromonas↗

Temperature-dependent protein and lipopolysaccharide expression in clinical Aeromonas isolates.

Clinical isolates of Aeromonas were grown at eight different temperatures from 10 degrees C to 40 degrees C. Whole cell lysates were examined by SDS-PAGE and major temperature-dependent changes to both protein and lipopolysaccharide (LPS) profiles were identified. Cells grown at the higher temperatures (37 degrees C and 40 degrees C) produced abundantly a protein of c. 60 kDa which was not detected at the lower temperatures. Temperature-dependent expressions of other proteins were also noted but these were more variable among the isolates. An effect of temperature on expression of lipopolysaccharides was also noted in that some strains produced significantly less O-polysaccharides at the higher temperatures. After fractionation of cells, major differences in the expression of cell envelope and outer-membrane proteins between cells grown at low and high temperatures were noted although no unifying patterns could be discerned. Such growth temperature-induced changes in the cell envelope constituents have not been described previously for Aeromonas isolates from man.

Aeromonas↗

Characterisation of a type IV pilus produced by Aeromonas caviae.

A pilus produced by a clinical isolate of Aeromonas caviae (strain CA195) was purified and partially characterised. The Mr of the pilin was estimated to be 23 kDa by SDS-PAGE. Its N-terminal amino-acid sequence showed that it was closely related to 'bundle-forming' type IV pili purified from other Aeromonas spp. associated with gastro-enteritis and considered to be important intestinal colonisation factors. Bundle-forming pili, often with a polar location, were seen on the surface of strain CA195 which was highly adherent to HEp-2 cells. Removal of surface structures by mechanical means decreased adhesion (by > or = 50%) suggesting that these pili played some role in HEp-2 cell binding. This pilus type could prove an important marker for enteropathogenic A. caviae which appear to lack other putative virulence factors.

Aeromonas↗

Electron microscopic examination of factors influencing the expression of filamentous surface structures on clinical and environmental isolates of Aeromonas veronii Biotype sobria.

Strains of Aeromonas veronii biotype sobria isolated from clinical and environmental sources were examined for their expression of surface structures under a variety of culture conditions. When grown on solid media at 37 degrees C, more than 95% of bacteria from the majority of strains isolated from human diarrheal feces and chicken carcasses were non-piliated or expressed only a few pili of long, flexible morphology per cell. Strains isolated from water or other foods were much more likely to express pili. Heavily piliated strains (all sources) possessed pili of several morphological types, including long, flexible pili of varying widths and rigid pili of varying lengths. Expression of Pili was favored by growth at temperatures ca. 20 degrees C and below and growth in liquid medium. Most fecal strains expressed some pili under these conditions. In addition, other surface structures (fibrillar aggregates, fibrillar networks bundle-forming pili) were seen on some strains from most sources. These were also seen most frequently when bacteria were grown in liquid media at temperatures ca. 20 degrees C and below. Pili expression was not dramatically influenced by growth under anaerobic conditions, or in iron-depleted media, or by combinations of the above conditions. The role of the above surface structures in Aeromonas pathogenicity remains to be elucidated.

Aeromonas↗

Niche-specific association of Aeromonas Ribotypes from human and environmental origin.

A total of 88 Aeromonas isolates from distinct locations and sources (39 from extraintestinal infections, 31 from diarrhoeic, ten from non-diarrhoeic faeces, all human, and eight from fresh water) were subjected to phenospecies identification, serotyping, ribotyping and detection of some virulence markers. The strains belonged to four different phenospecies marked by 19 O serogroups and 38 ribotypes. No strong correlation between these parameters was found, and no group, as defined by the typing methods, could be characterized with a particular set of virulence markers. There was a clear association of ribotypes with the source of the strains. Cluster analysis allowed the identification of a complex of ribotypes belonging to distinct but related sources, including clinical and environmental isolates. These results suggest that ribotyping may be an epidemiological tool suitable for the study of Aeromonas infections.

Aeromonas↗

Ampicillin-dextrin agar medium for the enumeration of Aeromonas species in water by membrane filtration.

Published selective media were evaluated for the isolation of Aeromonas spp. from environmental samples by membrane filtration. Satisfactory recoveries were obtained only with mA agar (Rippey & Cabelli) and dextrin-fuchsin-sulphite agar (Schubert), but neither was sufficiently selective. The positive aspects of these two media were combined in a new medium, ampicillin-dextrin agar. Recovery from pure cultures and environmental samples was optimal at an ampicillin concentration of 10 mg/l and incubation for 24 h at 30 degrees C under aerobic conditions, and specificity was high (i.e. confirmation rate usually greater than 90%, no false negative colonies encountered). The medium can also be used for isolation of Aeromonas spp. from sea water provided that the vibriostatic agent 0/129 is added at 50 mg/l.

Aeromonas↗

A note on Aeromonas spp. from chickens as possible food-borne pathogens.

The possible role of Aeromonas spp. as potential food-borne psychrotrophic pathogens was investigated by examining organisms isolated from processed raw chicken for their biochemical characteristics, ability to produce exotoxins and to grow at chill temperatures. These strains, in particular A. sobria, with identical characteristics to human diarrhoea-associated aeromonads were readily found. Chicken, and human and environmental (water) strains characterized in a previous study, were investigated for their ability to grow at refrigeration temperatures (5 +/- 2 degrees C) and, for selected strains, the theoretical minimum temperature for growth (Tmin) was determined from the growth pattern in a temperature gradient incubator. All enterotoxigenic chicken strains tested were typical mesophiles, with an optimal growth temperature of approximately 37 degrees C and Tmin values approximately 4.5 degrees C. They were rapidly outgrown by a psychrotrophic Pseudomonas sp. typical of spoilage biota found on food. Enterotoxin was not produced below 15 degrees C by any of the toxigenic food strains tested. The Aeromonas strains isolated from chickens in this study seem unlikely therefore to be a significant health risk, provided the chickens are properly stored and cooked. This would appear to be substantiated by the lack of reports of food-associated outbreaks of illness from these sources.

Aeromonas↗

Characterization of Aeromonas salmonicida subsp. salmonicida: a comparative study of strains of different geographic origin.

A total of 130 strains of the fish pathogen Aeromonas salmonicida isolated in Denmark, Norway, Scotland, Canada and the USA were examined. The strains originated from farmed salmonid fish. The biochemical, physiological and serological characteristics, antibiotic resistance patterns and cell surface-related properties were compared. Aeromonas salmonicida was found to be remarkably consistent in general cultural and biochemical characteristics. It is noteworthy that the strains were positive in the fermentation of L-arabinose and were negative in the fermentation of D-arabinose. All the strains were highly proteolytic. It was observed, however, that 5% of the strains did not digest calf and trout serum and the production of haemolysin and degradation of casein by the same strains were delayed compared with the other strains. Common to all of the rough strains were auto-aggregation and ability to bind the dyes Coomassie brilliant blue and Congo red and the majority of these strains were highly hydrophobic. The strains were tested for their susceptibility to 22 antibacterial agents. Antibiotic resistance profiles of Aer. salmonicida indicated that resistance to the quinolones and oxytetracycline was increasing and that multi-resistant strains were found in several countries. The variation found in antibiograms could have potential as epidemiological markers in certain geographic areas.

Aeromonas↗

Diversity of Aeromonas sp. in Flemish drinking water production plants as determined by gas-liquid chromatographic analysis of cellular fatty acid methyl esters (FAMEs).

Gas-liquid chromatography of cellular fatty acid methyl esters (FAMEs) was used to determine the phenotypic and genotypic diversity among 489 presumptive Aeromonas strains isolated from five Flemish drinking water production plants. FAME profiles were compared with the predetermined library profiles of a representative database, AER48C, which contains the mean FAME data of all 14 currently established hybridization groups (HGs) or genospecies within Aeromonas. Using AER48C, more than 93% (457 strains) of all presumptive aeromonads isolated on ampicillin-dextrin agar were unequivocally identified as belonging to this genus. Moreover, 85.5% and 73.5% of these strains could be assigned to a particular phenospecies or HG, respectively. Raw and treated surface water samples were dominated by members of the Aer. hydrophila complex (38.8%, comprising HGs 1-3), followed by the Aer. caviae complex (22.7%, comprising HGs 4-6) and the Aer. sobria complex (16.7%, comprising HGs 7-9). HGs 3, 5A/B and 8 were the most prominent genospecies in this type of water. On the other hand, it was found that raw and treated phreatic groundwater samples displayed a much more limited species diversity since these were almost entirely dominated (95.8%) by strains belonging to HGs 2 and 3 of the Aer. hydrophila complex. In general, flocculation-decantation and sand filtration were not shown to influence the overall species distribution in any of the plants examined.

Aeromonas↗

Survival potential of Aeromonas hydrophila in freshwaters and nutrient-poor waters in comparison with other bacteria.

The survival of a genetically-marked Aeromonas hydrophila strain was studied in water microcosms using viable counts. Aeromonas hydrophila AWWX1 was shown to survive without decline in viable counts for at least 10 d in three of four filtered-autoclaved freshwaters (surface water and groundwater) and in all examined filtered-autoclaved nutrient-poor waters (bottled spring water, Milli-Q and tap water). However, in the unfiltered waters, a rapid decrease in viable counts of Aer. hydrophila AWWX1 was observed after 1-5 d. The survival of Aer. hydrophila AWWX1 in nutrient-poor waters was compared with that of Pseudomonas fluorescens P17 and Spirillum strain NOX. Survival characteristics were organism- and water-dependent. In the filtered-autoclaved waters, viable counts of Spirillum strain NOX were ca 1 log-unit higher than for Aer. hydrophila AWWX1 and Ps. fluorescens P17. The tested strains Aer. hydrophila AWWX1 and Ps. fluorescens P17 survived 3 to 20, respectively 2 to 4 times better in the filtered-autoclaved waters compared to the unfiltered waters. Apparently, any inherent capability of these micro-organisms to adapt to low-nutrient environments was undone by the presence of the autochthonous microbiota. The present findings that Aer. hydrophila survives very poorly in several drinking waters is of utmost importance towards public health and arises questions about the mechanisms involved.

Aeromonas hydrophila↗

Pulsed-field gel electrophoresis as an epidemiological tool for clonal identification of Aeromonas hydrophila.

Pulsed-field gel electrophoresis (PFGE) was used to characterize Aeromonas hydrophila strains isolated from a cluster of hospital-acquired infections that occurred over approximately 1 month in a French hospital. Five isolates from patients and 10 isolates from the water supply were characterized by biotyping and antibiotic susceptibility patterns and compared with 10 epidemiologically unrelated strains isolated from patients and rivers, by PFGE of digests of chromosomal DNA. Five environmental and four clinical isolates belonged to the same biotype and antibiotic susceptibility pattern type. The endonucleases XbaI, SpeI and SwaI gave satisfactory profiles whereas DraI did not. The profiles were stable, reproducible and discriminatory. The 10 epidemiologically unrelated strains exhibited 10 different patterns after digestion with XbaI, the least expensive, suitable endonuclease. PFGE is a rapid and discriminatory technique for the typing of Aeromonas hydrophila where a common origin of infection is suspected.

Aeromonas hydrophila↗

Adhesive properties of a LamB-like outer-membrane protein and its contribution to Aeromonas veronii adhesion.

AIMS: To identify and characterize nonfimbrial proteins from Aeromonas veronii involved in the attachment to epithelial cells in vitro. METHODS AND RESULTS: Two Aer. veronii mucin- and lactoferrin-binding proteins with molecular masses of 37 and 48 kDa were identified by Western blot analysis. According to its N-terminal amino acid sequence, the 48-kDa protein was identified as Omp48, an outer-membrane protein similar to LamB of Escherichia coli. LamB is a well-known porin involved in maltose transport across the outer membrane in E. coli. In a microtitre plate assay, Omp48 bound to the immobilized extracellular matrix proteins collagen and fibronectin, and the mucin- and lactoferrin-binding activity was confirmed. Adhesion of Omp48 to mucin, lactoferrin and collagen was diminished by preincubation with homologous glycoproteins or other carbohydrates, suggesting a putative Omp48 lectin-like binding domain. Anti-Omp48 antiserum significantly inhibited the Aer. veronii adhesion to confluent HeLa cell monolayers and pretreatment of cells with purified Omp48 elicited competitive inhibition of adhesion. Similarly, cross-inhibition of Aer. hydrophila and Aer. caviae adhesion was achieved with the same treatments, indicating the existence of a conserved surface protein among these species. CONCLUSIONS: Taken together, these data indicate that Omp48 is involved in Aer. veronii adhesion to epithelial cells and might be an alternative adhesion factor of this micro-organism. SIGNIFICANCE AND IMPACT OF THE STUDY: The adhesive potential of Aeromonas spp. is correlated with pathogenicity; however, the adhesion mechanism is complex and not well understood. This study provides evidence of a putative adhesion factor that might be contributing to pathogenicity of Aer. veronii and could be used for vaccine development.

Aeromonas↗

Maintenance of pathogenicity during entry into and resuscitation from viable but nonculturable state in Aeromonas hydrophila exposed to natural seawater at low temperature.

AIMS: To investigate the fate of Aeromonas hydrophila pathogenicity when cells switch, in nutrient-poor filtered sterilized seawater, between the culturable and nonculturable state. METHODS AND RESULTS: Aeromonas hydrophila ATCC 7966, rendered non culturable within 50-55 days of exposure to marine stress conditions, was tested for its ability to maintain haemolysin and to adhere to McCoy cells. Results showed that pathogenicity was lost concomitantly with culturability, whereas cell viability remained undamaged, as determined by the Kogure cell elongation test. However, this loss is only temporary because, following temperature shift from 5 to 23 degrees C, multiple biological activities of recovered Aer. hydrophila cells, which include their ability to lyse human erythrocytes and to attach and destroy McCoy cells were regained. During the temperature-induced resuscitation, constant total cell counts were observed. Moreover, no significant improvement in recovery yield was obtained on brain-heart infusion (BHI) agar plates amended with catalase. We suggest that in addition to the growth of the few undetected culturable cells, there is repair and growth of some mildly injured viable but nonculturable cells. CONCLUSIONS: The possibility that nonculturable cells of normally culturable Aer. hydrophila in natural marine environment may constitute a source of infectious diseases posing a public health problem was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments may mimic what happens when Aer. hydrophila cells are released in natural seawater with careful attention to the conditions in which surrounding waters gradually become warmer in late summer/early autumn.

Aeromonas hydrophila↗

Aeromonas sobria, a causative agent of disease in farmed perch, Perca fluviatilis L.

Significant numbers of perch, Perca fluviatilis, raised on a pilot fish farm in Switzerland presented focal skin lesions on the lateral sides and fin rot. Mortality rates reached levels of up to 1% of the total fish on the farm per day. Virtually pure cultures of Aeromonas sobria were isolated from the liver, kidney, spleen and skin lesions of affected fish. Aeromonas sobria isolated from the farmed perch had a haemolytic effect on sheep and trout erythrocytes, autoaggregated, was cytotoxic for cultured fish cells and possessed genes involved in type III protein secretion. Experimental infection of naive perch with a single colony isolate of A. sobria from an affected farm fish resulted in the development of clinical signs identical to those seen on the farm. The results indicate that A. sobria can act as a primary pathogen of perch.

Aeromonas↗